Multiplexed mapping of the interactome of GPCRs with receptor activity–modifying proteins

Receptor activity–modifying proteins (RAMPs) form complexes with G protein–coupled receptors (GPCRs) and may regulate their cellular trafficking and pharmacology. RAMP interactions have been identified for about 50 GPCRs, but only a few GPCR-RAMP complexes have been studied in detail. To elucidate a comprehensive GPCR-RAMP interactome, we created a library of 215 dual epitope-tagged (DuET) GPCRs representing all GPCR subfamilies and coexpressed each GPCR with each of the three RAMPs. Screening the GPCR-RAMP pairs with customized multiplexed suspension bead array (SBA) immunoassays, we identified 122 GPCRs that showed strong evidence for interaction with at least one RAMP. We screened for interactions in three cell lines and found 23 endogenously expressed GPCRs that formed complexes with RAMPs. Mapping the GPCR-RAMP interactome expands the current system-wide functional characterization of RAMP-interacting GPCRs to inform the design of selective therapeutics targeting GPCR-RAMP complexes.


Web interface to browse the GPCR-RAMP interactome.
The results from the GPCR-RAMP interactome analysis are available in an R-based Shiny app that can be accessed in a web browser.The web interface presents a summary of GPCR-RAMP interactions detected for all possible capture-detection schemes.(A) The display for the adenosine A1 receptor (ADORA1) is presented as an example.Results are displayed as a binary heatmap and alluvial plot, and the density plots for the GPCR-specific capture of GPCR-RAMP complexes with anti-GPCR Human Protein Atlas (HPA) Abs are shown.A similar display is available on the app for each GPCR tested.(B) The app allows the user to customize the analysis of the GPCR-RAMP interactome dataset to create heatmap displays.Reproducibility of the SBA method to detect GPCR-RAMP complexes.
The reproducibility of the SBA assay for detecting solubilized GPCR-RAMP complexes was tested.Representative examples of GPCRs were selected from two subfamilies.The GPCRs were expressed with or without a RAMP in biological triplicate, and each sample was quantified in technical duplicate (N=6).GPCR class C group 5 member A (GPRC5A; glutamate subfamily) was expressed with or without RAMP2 (A), and orexin receptor type 2 (HCRTR2; beta subfamily) was expressed with or without RAMP3 (B).Each complex was detected using four epitope-based capture-detection strategies in parallel.Top row: the RAMP was captured with beads coupled to anti-HA or anti-OLLAS mAb, and the GPCR was detected with PE-conjugated anti-1D4 mAb.Bottom row: the GPCR was captured with beads coupled to anti-1D4 or anti-FLAG mAb, and the RAMP was detected with PE-conjugated anti-OLLAS mAb.Significance was determined by a one-sided unpaired Wilcoxon test (**** p < 0.0001, ** p < 0.01).Sample sizes and p-values are listed in Table S3.X-axis labeling: mock indicates that only the GPCR was ectopically expressed; RAMP2 (A) or RAMP3 (B) indicate that the GPCR was coexpressed with the specified RAMP.Data are plotted as the log2 of median fluorescence intensity (MFI), and horizontal bars represent the medians of each group with the 25 th and 75 th percentiles.Capture RAMP1 complex was detected with PE-conjugated anti-1D4 mAb.CALCRL was captured with beads coupled to anti-CALCRL pAb, anti-FLAG mAb or anti-1D4 mAb, and the CALCRL-RAMP1 complex was detected with PE-conjugated anti-OLLAS mAb.Statistical significance was determined by ordinary one-way ANOVA followed by Dunnett's multiple comparisons test to mock.mock (**** p < 0.0001, if not marked p >= 0.05).The thick horizontal lines represent the median values, and the thin lines above and below show the 75th and 25th percentiles, respectively.Sample names on the x-axis use the format "transfected GPCR name (if any).transfectedRAMP name (if any)".Data are plotted as the log2 of median fluorescence intensity (MFI).Sample sizes and p-values are listed in Table S3.
reported in the literature is overlaid on respective cells as a solid black outline (interaction expected), a thin dashed outline (interaction not expected) or a thick dashed outline (interaction uncertain due to conflicting reports).White boxes indicate GPCR-RAMP pairs not tested due to preparation error.Green squares, complex detected; orange squares, complex not detected.validated anti-CALCRL HPA Abs.The complexes were detected using PE-conjugated anti-OLLAS mAb (top row) or PE-conjugated anti-RAMP1, anti-RAMP2 or anti-RAMP3 pAb (remaining rows).Statistical significance was determined by ordinary oneway ANOVA followed by Dunnett's multiple comparisons test to mock (i.e., CALCRL expressed alone) (**** p < 0.0001, * p < 0.01, if not marked p >= 0.05).Sample names on the x-axis denote transfected RAMP name (if any).Data plotted as signal-to-noise ratio (unitless) on a log2 scale.Signal-to-noise ratios: dashed line, 1; dotted line, 2. Sample sizes and p-values are listed in Table S3.

Detection of native RAMP expression in three cell lines.
Samples of solubilized lysates from Expi293F (Expi), SH-SY5Y and SK-N-MC cells were incubated with the SBA, which could capture native RAMP1, RAMP2 and RAMP3 with anti-RAMP specific Abs (Ab names are given above each plots).The proteins were detected by PE-conjugated anti-RAMP1, anti-RAMP2 or anti-RAMP3 pAbs.

Analysis of GPCR expression in human cells parsed by RAMP interaction.
(A) Left, Matrix showing the assignment of GPCR interaction with a particular RAMP as either yes (green), no (orange), or inconclusive (grey) based on the evidence class of the results from each of the two GPCR-RAMP complex capture schemes: epitope-based or

RNA expression of GPCRs and RAMPs in human cells and tissues.
(A) Violin plots showing GPCR-RAMP RNA expression ratios in human cells.GPCRs were annotated as RAMP-interacting or not based on the SBA assay results.The expression ratio between each GPCR and each RAMP was calculated across all human cell types with available RNASeq data.Cell types in which either GPCR or RAMP expression was less than 1 nTPM were excluded.The data are plotted as log2 of the GPCR-RAMP expression ratio and the number of datapoints for each dataset is provided at the bottom of each plot.S3).There were data on G protein coupling for 58 GPCRs and data for both G protein and b-arrestin coupling data for 32 GPCRs (n = 90 in total) out of the 215 GPCRs studied in this paper.All data are from gpcrdb.org(23)(24)(25)(26).
Table S1.Antibodies and GPCRs.List of all used antibodies (Ab), their target GPCR or RAMP, and information about each GPCR.Information about which assay each Ab was used in is also specified.
(provided as a separate Excel file) Table S2.
Fitting parameters of IP1 Dose-response curves.The values provided correspond to the dose-response curves presented in Fig. S2, computed in Prism 10.Curves were fit to a log[agonist] versus response three-parameter non-linear model with least squares fit at an alpha of 0.05.Df (degrees of freedom) was computed from the number of samples and groups and indicates the number of independent pieces of information.All other values presented are the calculated best-fit values.EC50, concentration of agonist that provokes a response halfway between the basal and maximum level of stimulation.
GPCRs without definitive evidence for interaction or noninteraction for each RAMP were excluded; 205 GPCRs out of 215 GPCRs were analyzed in total.Significance was determined by a two-sided unpaired Wilcoxon test (** p < 0.005, p >= 0.05 if not marked).Sample sizes and p-values are listed in TableS3.(B)Binaryheatmaps are shown for RAMP expression, where each row shows data from a human tissue type (left) or single-cell data from a human cell type (right).Green, RAMP expression greater than 1 nTPM.Purple, RAMP expression less than 1 nTPM.(C)Heatmap is shown based on single-cell data for RAMP expression in different human cell types (rows) for each RAMP (columns) arranged by hierarchical clustering.Data are from proteinatlas.org(21,22).one in five GPCRs couples to more than one G protein subtype.(C) GPCR coupling to barrestin subtypes based on published log(Emax/E50) values, assigned as no coupling if 0 and coupling if > 0. Significance was determined by the Fisher exact test (* p < 0.05, p >= 0.05 if not marked, Table evidence for RAMP interaction are classified as "RAMP Interaction" and those that do not are classified as "No RAMP Interaction".G protein subtype is color-coded and G protein coupling for a given GPCR is classified as either primary or secondary.(B) The vast majority of GPCRs couple to a single G protein subtype.However, approximately